The methylotrophic yeast Pichia pastoris has emerged as a prominent host for the industrial production of recombinant proteins due to its unique biological characteristics and well-developed expression systems. In light of the increasing economic importance of large-scale recombinant protein production, non-conventional yeasts such as P. pastoris are garnering significant attention for their ability to produce high yields of target proteins. This study focuses on the development and optimization of an innovative recombinant expression strategy in P. pastoris for the high-level bench-scale production of extracellular lipases. Three candidate lipases- Yarrowia lipolytica lipase (YlLip2), Candida antarctica lipase B (CALB), and Thermomyces dupontii lipase (TDL) - were cloned and expressed using tailored expression constructs. The produced lipases were systematically evaluated across a range of pH and temperature conditions to determine their reaction rates, substrate specificity, and stability. Following production, the enzymes were purified and characterized to assess their potential for future applications in biocatalysis. The results demonstrate that the optimized P. pastoris expression system is capable of generating high levels of functional extracellular lipases, paving the way for further investigations into their industrial and environmental applications.
